1. Field of the Invention
The present invention is broadly concerned with methods for synthesizing various epothilone segments or precursors (either naturally occurring or analogs thereof) which can be used for the efficient synthesis of complete epothilones.
2. Description of the Prior Art
The epothilones (16-membered macrolides which were initially isolated from the myxobacterium Sorangium cellulosum) represent a class of promising anti-tumor agents, and have been found to be potent against various cancer lines, including breast cancer cell lines. These agents have the same biological mechanism of action as Taxol, an anti-cancer drug currently used as a primary therapy for the treatment of breast cancer. Other potential applications of the epothilones could be in the treatment of Alzheimer""s disease, malaria and diseases caused by gram-negative organisms. Other cancers such as ovarian, stomach, colon, head and neck and leukemia could also potentially be treated. The epothilones also may have application in the treatment of arthritis.
In comparison to Taxol(copyright), the epothilones have the advantage of being active against drug-resistant cell lines. Drug resistance is a major problem in chemotherapy and agents such as the epothilones have overcome this problem and hold great promise as effective agents in the fight against cancer.
In addition, the poor water solubility of Taxol(copyright) has led to the formulation of this drug as a 1:1 ethanol-Cremophor concentrate. It has been determined that the various hypersensitive reactions in patients such as difficulty in breathing, itchiness of the skin and low blood-pressure are caused by the oil Cremophor used in the formulation. The epothilones are more water soluble than Taxol(copyright) which has positive implications in its formulation. Further advantages of the epothilones include easy access to multi-gram quantities through fermentation procedures. Also the epothilones are synthetically less complex, thus structural modifications for structure activity relationship studies are easily accessible.
The epothilones exhibit their activity by disrupting uncontrolled cell division (mitosis), a characteristic of cancer, by binding to organelles called microtubules that are essential for this process. Microtubules play an important role in cell replication and disturbing the dynamics of this component in the cell stops cell reproduction and the growth of the tumor. Antitumor agents that act on the microtubule cytoskeleton fall into two general groups: (1) a group that inhibits microtubule formation and depolymerizes microtubules and, (2) a group that promotes microtubule formation and stabilizes microtubules against depolymerization. The epothilones belong to the second group and have displayed cytotoxicity and antimitotic activity against various tumor cell lines.
It has been demonstrated on the basis of in vitro studies that the epothilones, especially epothilone B, are much more effective than Taxol(copyright) against the multi-drug resistant cell line KBV-1. Preliminary in vivo comparisons with Taxol(copyright) in CB-17 SCID mice bearing drug-resistant human CCRF-CEM/VBL xenografts have shown that the reduction in tumor size was substantially greater with epothilone B in comparison to Taxol(copyright).
In light of the great potential of the epothilones as chemotherapeutic agents, there is a need for techniques allowing the practical, large scale, economical synthesis thereof. Furthermore, there is a need for synthetic methods which facilitate the preparation of various homologs and analogs of the known epothilones, and those having affinity labels allowing study of the binding interactions of these molecules.
The present invention overcomes the problems outlined above, and provides various practical, commercially feasible synthetic routes for the production of important epothilone precursors or segments in high yield. The invention is particularly concerned with the synthesis of the precursors or segments C, D (which is a combination of segments B and C) and vinyl halide epothilone precursors.
In a first aspect of the invention, a C1-C6 segment C epothilone precursor of the formula 
is synthesized using a Noyori reduction reaction. In the foregoing formula, n1 is an integer from 0-4, R4 is selected from the group consisting of H, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups, R5 and R6 are each individually and respectively selected from the group consisting of H, substituted and unsubstituted aryl and heterocyclic groups, C1-C10 straight and branched chain alkyl groups, and substituted and unsubstituted benzyl groups, R7 is H or straight or branched chain C1-C10 alkyl groups, and Pxe2x80x2 is a protective group (as used herein, Pxe2x80x2 is any suitable protective group, and where more than one Pxe2x80x2 is in a single formula, the protective group may be the same or different).
The method comprises the steps of first providing a xcex2-keto ester of the formula 
where n1, R5, R6, R7 and Pxe2x80x2 are as defined above, and T is an alkyl group. This xcex2-keto ester is then preferentially hydrogenated at the C3 keto group to form the corresponding hydroxyester. This is accomplished by reacting the xcex2-keto ester with a hydrogenating agent in the presence of an asymmetric organometallic molecular catalyst comprising a metal atom or ion having one or more chiral ligands coupled thereto. The synthesis is completed by then converting the hydroxyester to the epothilone precursor.
More preferably, n1is an integer from 0-4, R5, R6 and R7 are each individually and respectively selected from the group consisting of H and the straight and branched chain C1-C4 lower alkyls, and the protective group is benzyl. In terms of preferred process parameters, the hydrogenating agent is preferably H2 and the hydrogenating step is carried out at a pressure of from about 30-100 psi, more preferably 50-75 psi, and at a temperature of from about 40-100xc2x0 C., more preferably from about 50-75xc2x0 C. The reaction is normally allowed to proceed for a period of from about 12 hours to 5 days, and more usually for about 2-5 days. Typically, the reaction mixture is agitated during the hydrogenating step.
The catalyst used in the hydrogenation reaction is preferably one of the well-known Noyori catalysts such as RuBr2(S)-binap. However, a variety of other catalysts of this type can also be employed. The catalyst is generally used at a level of from about 1-25 mol % in the reaction mixture.
In order to complete the reaction sequence, the hydroxyester resulting from the Noyori reduction is converted to the epothilone precursor segment C. A number of routes can be used to effect this conversion. Preferably, however, the conversion involves: (1) removing the Pxe2x80x2 protecting group from the hydroxyester to form a diol; (2) protecting the oxygen atoms of the diol, forming a protected diol; (3) reducing the ester function of the protected diol to a primary alcohol; (4) oxidizing the primary alcohol to the corresponding aldehyde; (5) reacting the aldehyde with a Grignard reagent having the R4 group coupled thereto to form a secondary alcohol; and (6) oxidizing the secondary alcohol to form the final epothilone precursor.
Preferably, the Pxe2x80x2 removal step involves reacting the hydroxyester with hydrogen in the presence of a catalyst (e.g., Pd(OH)2 or Pd/C) at a pressure of from about 40-100 psi. The oxygen atom protecting step comprises reacting the diol with TBS chloride in a compatible solvent (i.e., one that will not interfere with the desired reaction) at a temperature of from about 40-100xc2x0 C. for a period of from about 30-60 hours. The ester function reduction step is preferably carried out by reacting the protected diol with the reducing agent DIBAL-H at a temperature of from about xe2x88x9220 to xe2x88x9285xc2x0 C. The oxidation of the primary alcohol is carried out most conveniently using 4-methylmorpholine N-oxide and a catalytic amount of tetrapropylammonium perruthenate. The Grignard reaction serving to attach the R4 group is entirely conventional and well within the skill of the art; likewise, the final oxidation of the secondary alcohol is trivial using the aforementioned oxidation procedure, i.e., NMO and TPAP.
The C1-C6 Formula I segment C can also be produced by a synthesis wherein a nitrile compound of the formula 
where Pxe2x80x2, R7 and n1, are as defined above and the value of each n1 may be the same or different, is alkylated to yield a dialkylated nitrile compound of the formula 
where Pxe2x80x2, R5, R6, R7, and n1 are as defined above and the value of n1 may be the same or different; and the dialkylated compound is then converted to the desired C1-C6 segment C epothilone precursor.
The converting step preferably involves oxidizing the dialkylated compound III to yield a ketone of the formula 
where Pxe2x80x2, R4, R5, R6, and n1 are as defined above and the value of each n1 may be the same or different, and converting the ketone to the C1-C6 epothilone precursor.
Alternately, the dialkylated nitrile compound defined above may be treated by deprotecting the nitrile to yield a diol compound having the formula 
where R5, R6, R7 and n1 are as defined above and the value of each n1 may be the same or different, and thereafter converting the diol compound to the C1-C6 epothilone precursor.
A still further synthesis of the Formula I C1-C6 segment C precursor comprises providing an ester compound of the formula 
where R5, R6, R7, Pxe2x80x2 and n1, and Rxe2x80x2 is a C1-C10 straight or branched chain alkyl group reacting the ester compound VIII with a sulfone to acylate the ester, and thereafter desulfonating the acylated ester to obtain the desired segment C epothilone precursor. The sulfone is preferably of the formula
X1xe2x80x94SO 2xe2x80x94R4xe2x80x83xe2x80x83VIII
where R4 is defined above and X1 is selected from the group consisting of unsubstituted aryl and heterocyclic groups. The most preferred sulfone is ethyl phenyl sulfone.
In another aspect of the invention, a method is provided for the production of D precursors, which are a combination of segments B and C. The segment C precursors are of course produced as outlined above. Segment B precursors are of the formula 
where n2 is an integer from 1-4, and R3 is selected from the group consisting of H, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups. This segment can be efficiently produced using known techniques.
The segments B and C are connected by first reacting the segment C precursor with a base to form an enolate, followed by reacting the enolate with the segment B. These reactions are generally carried out by initially cooling the base to a temperature of about xe2x88x9275xc2x0 C., adding the segment C precursor and elevating the temperature of the mixture to about xe2x88x9240xc2x0 C., then recooling the mixture to at least about xe2x88x9275xc2x0 C. and adding the precursor segment B thereto.
The invention also is concerned with a method of synthesizing vinyl halide epothilone precursors having the general formula 
where n3 is an integer from 1-4, R is selected from the group consisting of C4-C8 cycloalkyl, and substituted and unsubstituted aromatic and heteroaromatic groups, R1 and R2 are each individually and respectively selected from the group consisting of H, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups, Pxe2x80x2 is a protecting group, and M is either bromine or iodine. This reaction involves first providing an alkynyl ketone of the formula 
wherein n3 and Pxe2x80x2 are as previously defined. Thereafter, the alkynyl ketone is asymmetrically reduced to create the alcohol form of the alkynyl ketone. This alcohol form is then reacted with a reagent system selected from the group consisting of (R1)3Al and zirconocene dichloride or stannyl cupration reagent and R1-halide to form a vinyl metal species. The vinyl metal species is then reacted with an aryl or vinyl halide to form an allyl alcohol. This allyl alcohol is then converted to the vinyl halide epothilone precursor.
Normally, the asymmetric reduction step involves creating the reduced form of the alkynyl ketone and the resulting alcohol is protected using TBS as a protecting agent. The R1-halide is selected from the group consisting of R1Br and R1I. The conversion step preferably includes the step of initially converting the allyl alcohol to an alkynyl stannane, reducing the stannane with chlorohydridozirconocene to form a 1,1-dimetallo Zrxe2x80x94Sn species. The dimetallo species is then hydrated to form a vinyl stannane, which is then quenched with either iodine or bromine. Alternately, the conversion step may be accomplished by transmetallating the dimetallo species with an organocuprate, quenching with an alkyl-R2-OTf, and final quenching with either iodine or bromine incorporating the R2 group.
Preferred vinyl halide C12-C20 epothilone precursors of the formula 
where R8 is selected from the group consisting of H, C1-C4 straight or branched chain alkyl, alkenyl or alkynyl groups, R9 is selected from the group consisting of H, C1-C10 straight and branched chain alkyl, alkenyl, alkynyl, hydroxyalkyl, hydroxyalkenyl or hydroxyalkynyl groups, substituted and unsubstituted cyclic, heteroxylic and aryl groups, R12 is selected from the group consisting of H, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups, X2 is O or S, n4 is an integer which ranges from 1 to 4, Pxe2x80x2 is a protective group and M is either iodine or bromine, may be produced as follows.
First, an alcohol of the formula 
where R8, R9, X2, n4 and Pxe2x80x2 are as defined above, is converted to the C12-C20 epothilone segment A. This method preferably comprises the steps of:
providing an enone compound of the formula 
xe2x80x83where R8, R9, X2, Pxe2x80x2 and n4 are as defined above, and asymmetrically reducing the enone compound XIV in the presence of a chiral catalyst to obtain the alcohol, compound XIII. The alcohol compound XIII is then protected at the C15 alcohol position, followed by known conversion steps to precursor Formula XII.
The enone compound XIV is preferably obtained by reacting in a basic reactive medium starting aldehyde compound of the formula 
where R9 and X2 are as defined above, with a phosphonate compound of the formula 
where R8, Pxe2x80x2 and n4 are as defined above, and R10 and R11 are individually selected from the group consisting of C1-C4 straight or branched chain alkyl groups. In particularly preferred forms, R8 and R9 are each H, X is S, n4 is 1, and Pxe2x80x2 is TBS. The chiral catalyst is preferably (R)-B-Me-CBS-oxazaborolidine.
A still further method of synthesizing the preferred C12-C20 epothilone precursors of Formula XII described immediately above involves conducting an aldol condensation reaction using an aldehyde with an enolate anion to give a xcex2-keto alcohol; this alcohol is then oxidized to the ester form followed by an asymmetric reduction to yield a chiral alcohol. Preferably, the method comprises providing an aldehyde of the formula 
where R8, R9 and X2 are as defined above, reacting this aldehyde with an acetate of the formula 
where R13 is a methyl group, Z is a C1-C4 straight or branched chain alkyl group or a substituted or unsubstituted benzyl group in a basic reaction mixture to yield a xcex2-hydroxyester of the formula 
where R8, R9, X2, and Z are as defined above.
The xcex2-hydroxyester is then oxidized to the corresponding xcex2-ketoester of the formula 
where R8, R9, X2, and Z are as defined above. Next, xcex2-ketoester is hydrogenated to form a chiral alcohol of the formula 
where R8, R9, X2, and Z are as defined above, by reacting the xcex2-ketoester with a hydrogenating agent in the presence of asymmetric organometallic molecular catalyst comprising a metal atom or ion having one or more chiral ligands coupled thereto. Finally, the chiral alcohol is converted to the C12-C20 epothilone of Formula XII.
In preferred forms, the acetate is ethyl acetate, and the aldehyde and acetate are reacted in the presence of an alkali metal diisopropyl amide in a solvent selected from the group consisting of THF, a mixture of t-butanol and t-butoxide, sodium ethoxide, and ethanol. The reaction temperature is preferably from about xe2x88x9250 to xe2x88x92125xc2x0 C. The xcex2-ketohydroxyester is preferably oxidized using an alkali metal or alkaline earth metal oxide or hydroxide. The hydrogenating step preferably uses hydrogen and is carried out at a pressure of from about 30-100 psi, and a temperature from about 40-100xc2x0 C.
The molecular architecture of the representative epothilones (Formulae A-B) reveals three essential domains. These include the two chiral domains, namely the C1-C8 polypropionate region and the C12-C15 region, and the achiral spacer C9-C11 which unites the chiral domains. Additional structural features include a thiazole moiety, the C16 double bond, a methyl group at C4 and a cis-epoxide moiety (C12-C13) in the epothilones of Formula A. In the following formulae A and B, n1 is an integer from 0-4, n2 and n3 are each respectively integers from 1-4, R is selected from the group consisting of C4-C8 cycloalkyl, and substituted and unsubstituted aromatic and heteroaromatic groups, R1, R2, R3 and R4 are each individually and respectively selected from the group consisting of H, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups, R5 and R6 are each individually and respectively selected from the group consisting of H, substituted and unsubstituted aryl and heterocyclic groups, C1-C10 straight and branched chain alkyl groups, and substituted and unsubstituted benzyl groups, R7 is H, or straight or branched chain C1-C10 alkyl groups, X is either oxygen or NH, and Y is either oxygen or H2. 
Scheme 1 below outlines a retrosynthetic analysis respecting the total synthesis of the epothilones of Formula A in accordance with the invention, where each n1 and n2 equal 1, R is 2-methyl-thiazol-4-yl, R1, is methyl, R2 is H or methyl, R3, R4, R5 and R6 are methyl, R7 is H, and X and Y are oxygen. Standard epoxidation and macrolactonization strategies are used for the formation of the C12-C13 epoxide moiety and the 16-membered macrolide. The analysis for other analog epothilones of Formula A is identical, and also for the epothilones of Formula B and its analogs, with the epoxidation step being omitted.
A novel route to a Formula I C1-C6 segment (labeled C in Scheme 1) utilizes a stereoselective hydrogenation reaction, i.e., a Noyori reduction. 
Synthesis of Segment C (C1-C6 of Formula A)
The invention makes it possible to synthesize several analogs of segment C as set forth in Formula I with various chain elongations and/or substitutions at C2 and substitutions at the xcex1-carbon relative to the keto group. It also allows for, as mentioned before, modifications at the carbon atom between the keto and the protected secondary hydroxy group with other groups. These chain extensions and substitutions are illustrated by a general Formula I, previously identified. The synthesis of these modified derivatives can be achieved utilizing chemistry exemplified in the synthesis of segment C in the Schemes described below. These modified segments can then be utilized in the total synthesis of various analogs of epothilones. 
The synthesis of the Forumula I segment has been accomplished via unique and complementary routes, detailed in Schemes 2 and 3 below, which illustrates the synthesis of the naturally occurring segment C. A novel step in the synthesis of the C1-C6 segment utilizes the Noyori hydrogenation of xcex2-keto ester 4 to generate the requisite stereochemistry at C3. This Noyori hydrogenation (Noyori, R. et al., Asymmetric Hydrogenation of, xcex2-Keto Carboxylic Esters. A Practical, Purely Chemical Access to xcex2-Hydroxy Esters in High Enantiomeric Excess, J. Am. Chem. Soc. 109:5856-5858 (1987)) provides the required enantiomer with high selectivities (92-95% enantiomeric excess). The use of a Noyori hydrogenation reaction permits large, commercial scale production of various segment C precursors.
The required xcex2-keto ester 4 is obtained in two steps from the readily available starting material 3-benzyloxypropionic acid (2). Asymmetric hydrogenation of 4 in methanol using RuBr2(S)-binap as catalyst at 60 psi gives the xcex2-hydroxyester 5 in 71-92% yield (92-95% ee). Deprotection of the benzyl ether and bis-silylation of the resultant diol 6 provides ester 7. The ester is reduced to the known primary alcohol 8 using DIBAL-H. The alcohol is then oxidized to the known aldehyde 9 using a previously unreported oxidation procedure. The aldehyde is then reacted with EtMgBr using a reported procedure (Claus, et al., Synthesis of the C1-C9 Segment of Epothilons, Tetrahedron Lett., 38:1359-1362 (1997)) to give the known secondary alcohol 10 in 65% yield. This alcohol is then oxidized to the C1-C6 segment C using TPAP and NMO.
In summary, although segment C is a key synthon in previously reported total syntheses (Nicolaou, et al., Total Syntheses of Epothilones A and B via a Macrolactonization-Based Strategy, J. Am. Chem. Soc., 119:7974-7991 (1997)) of the epothilones, the synthetic route utilizing the asymmetric Noyori hydrogenation is unique.
The alternate route toward segment C precursors allows for the introduction of affinity labels and modifications at the C4 position as shown in Scheme 3. Applying the Noyori reduction to the known unsubstituted xcex2-keto ester 11 provides a building block that can be used for the modifications at C4 of the epothilones. This Scheme accordingly allows for modification of the epothilones and gives a more general route to introduce a variety of substituents at this position.
Thus, the Noyori hydrogenation of xcex2-keto ester 11 yields the known xcex2-hydroxy ester 12 (Ali, et al., Formal Syntheses of Cryptophycin 1 and Arenastatin A, Tetrahedron Lett., 38:1703-1706 (1997)) in 97% yield (in 97% enantiomeric excess). The Frater alkylation of xcex2-hydroxy ester 12 yields the previously reported xcex1-methyl analogue 13 (Ali, et al., Formal Syntheses of Cryptophycin 1 and Arenastatin A, Tetrahedron Lett., 38:1703-1706 (1997)) in 71% yield (98% diastereomeric excess). A second Frater alkylation of hydroxyester 13 gave bis-dimethyl derivative 5 in 59% yield which was then converted to epothilone segment C by the chemistry shown in Scheme 2. At this stage, other substituents such as benzyl, allyl and other C1-C6 alkyl groups can be introduced by using other electrophiles in the second Frater alkylation in place of iodomethane. The novel aspect about this alternate route to segment C is the ability to alter the substituents at the C4 position of the epothilones using the aforementioned Frater alkylation strategy. 
In another aspect of the invention, the synthesis of exemplary segment C (and of course all of the other segment C analog precursors of Formula I) utilizes a starting material which can be obtained from lactose or malic acid and circumvents the need to construct the C3 stereochemistry using an asymmetric synthesis. This technique gives access to the C1-C6 segment of the epothilones by a concise route set forth in Scheme 3A. 
The Scheme 3A synthesis employs ethyl-(R)-4-cyano-3-hydroxybutanoate 12 as starting material. Selective reduction of the ester functionality using sodium borohydride in ethanol from 0xc2x0 C. to room temperature overnight gave (S)-3,5-dihydroxyvaleronitrile 13.
The product 13 was then protected at its free 4-hydroxyl group as a p-methoxybenzyl ether by forming a dibutyl tin acetal with dibutyltin dimethoxide in refluxing benzene, followed by treatment with p-methoxybenzyl chloride and tetrabutylammonium iodide at 60xc2x0 C. to give the primary ether derivative 14 in 61% yield.
The nitrile 14 is then alkylated using LDA and methyl iodide. The enolate of the nitrile generated using LDA is warmed to 60xc2x0 C. before the addition of methyl iodide to ensure dialkylation to give (S)-2,2-dimethyl-3-hydroxy-5-p-methoxybenzyloxy-valeronitrile 15 in 76% yield.
The dialkylated product 15 thus obtained is then refluxed with ethyl magnesium bromide in a THF solution with a catalytic quantity of copper bromide-dimethylsulfide complex and the resulting imine hydrolyzed in situ with 0.5 N aqueous citric acid solution for 5 hrs to give ketone 16. Deprotection of the PMB group on the ketone 16 with ceric ammonium nitrate with a 1:9 water:acetonitrile solvent mixture followed by protection of the diol with TBSOTf and 2,6-lutidine gave the ketone 18 which constitutes segment C, the C1-C6 carbon skeleton of the epothilones.
An even more preferred synthesis of the C1-C6 segment C precursors is a two-step, one-pot conversion of an intermediate methylester to the ethyl ketone using a sulfone anion to acylate the ester, followed by desulfonylation to provide segment C using sodium amalgam. This one-pot conversion achieves 90% yield and shortens the synthesis significantly. This preferred synthesis is set forth in the following Scheme 3B; again this scheme may be readily modified to obtain desired analogs defined by Formula I. 
Synthesis of Segment B (C7-C11 of Formula A)
The synthesis of the C7-C11 segment B is preferably achieved using previously reported chemistry (Lin, Efficient Total Syntheses of Pumiliotoxins A and B, Applications of Iodide-Promoted Iminium Ion-Alkyne Cyclization in Alkaloid Construction, J. Am. Chem. Soc., 118:9062-9072(1996)) and is outlined in exemplary Scheme 4, which is precursor of a naturally occurring epothilone. 
This synthesis can also be used to introduce various chain-elongations on this segment and to introduce various other substituents at C-8. These modifications can be illustrated by Formula IX (Segment D), wherein n2 and R3 are as defined previously. Their synthesis can be achieved using chemistry exemplified in the synthesis of segment B in Scheme 4. Again, these modified segments can then be utilized in the total synthesis of various analogs of epothilones. 
Synthesis of Segment D (C1-C11 of Formula A) via Aldol Reaction
The connection of the two segments C and B utilizes a highly diastereoselective aldol reaction, exemplified in Scheme 5 showing the connection of the two precursors B and C of a naturally occurring epothilone. When the C1-C6 ketone segment C is treated with abase, for example lithium diisopropylamide and the resultant enolate reacted with C7-C11 aldehyde segment B, a single desired diastereomer 14 was observed in 65% yield. This diastereselectivity is believed to arise from a favorable nonbonding interaction between the C10-C11 double bond and the carbonyl group of the aldehyde that gives rise to the desired diastereomer. After the connection is made, the resultant secondary alcohol is protected as the corresponding tert-butyldimethylsilyl ether.
Similar chemistries would apply for the connection of modified segments C and B of the type discussed previously and emplified by Formulae C and D. 
Proposed Synthesis of Segment A (C12-C20 of Formula A)
The invention also provides a new route to the C12-C20 segment (segment A of the naturally occurring epothilone), and corresponding analogs thereof. This involves new ways to set the C16-C17 trisubstituted double bond and the C12-C13 cis-double bond, which serves as precursor to the cis-epoxide at C12-C13 in the epothilones.
Stereoselective Construction of C16-C17 of Trisubstituted Olefin and Introduction of Thiazole in Formula A
The introduction of the thiazole moiety draws upon zirconium-catalyzed carboalumination chemistry (Wipf, Rapid Carboalumination of Alkynes in the Presence of Water, Agnew. Chem., Int. Ed. Engl., 32:1068-1071 (1993)) wherein a C16-C17 alkyne bond in an appropriately functionalized C13-C17 propargylic alcohol 16 (Scheme 6) is subjected to methylalumination in the presence of zirconocene dichloride (Cp2ZrCl2). The resultant alkenylalane is coupled with 2-methyl-4-bromothiazole 17 in the presence of zinc chloride under Pd(0) catalysis to access the trisubstituted E-olefin 19 stereoselectively following the protection of the alcohol 18 as the OTBS-ether.
The chiral propargylic alcohol 16 is obtained via the asymmetric reduction of the readily available alkynyl ketone 15. This is exemplified in Scheme 6, which illustrates the synthesis of the precursor for the naturally occurring epothilone. After the introduction of the thiazole moiety, the known primary alcohol 21 is revealed by deprotection of the PMB ether 19 and then oxidized to the previously reported (Mulzer, J., et al. Easy Access to the Epothilone Familyxe2x80x94Synthesis of Epothilone B, Tetrahedron Lett., 39:8633-8636 (1998)), C13-C20 aldehyde 22. 
Alternately, a stannylcupration-methylation methodology (Harris, et al., Synthetic Approaches to Rapamycin. 3. Synthesis of a C1-C21 Fragment, Synlett, pp. 903-905 (1996)) can be used in order to introduce the trisubstituted olefin. Thus the O-TBS ether 16a (Scheme 7) of propargylic alcohol 16 on treatment with the stannylcuprate reagent 20 followed by methylation with iodomethane provides the corresponding stannane which is then coupled under Stille conditions with the bromothiazole 17 to yield the olefin 19. 
The synthesis of 2-methyl-4-bromothiazole 17 from the known 2,4-dibromothiazole (Reynaud, et al., Sur une Nouvelle Synthese du Cycle Thiazolique, Bull. Soc. Chim. Fr., 295:1735-1738(1962)) is outlined in Scheme 8. 
The zirconium-catalyzed methylalumination strategy constitutes a novel route to construct the C16-C17 double bond and to introduce the thiazole ring. The novelty lies in the use of a chiral propargylic alcohol like 16 in the carbometalation reaction followed by the direct introduction of the thiazole unit.
This methodology also allows for the introduction of various substituents and chain elongations on the C12-C20 segment A. Thus starting with analogs of the ketone 15 in Scheme 6, a variety of chain-elongated derivatives of segment A can be produced. Also carrying out an ethylalumination (Et3Al) in place of methylalumination (AlMe3) (Scheme 6) allows the introduction of an ethyl group (Et) at C16. In the same context, other groups can also be introduced using the alternate stannylcupration-alkylation method by replacing iodomethane with other electrophiles in this reaction shown in Scheme 7. In addition, the thiazole ring can be replaced by other cyclic, aromatic and heteroaromatic rings by using other vinyl or aromatic/heteroaromatic halides in place of 2-methyl-4-bromothiazole 17 in the coupling reaction following either the carboalumination or stannylcupration strategy exemplified in Schemes 6 and 7 respectively.
Stereoselective Construction of the C12-C13 cis-olefinic Bond of Formula A
The goals in the construction of the C12-C13 Z-olefinic bond, were to design a method providing maximum control over the olefin geometry and to furnish common intermediates in the synthesis of both epothilones A and B. The introduction of affinity labels at C-12 was also a consideration.
The C12-C13 olefin can be constructed in the form of Z-vinyl iodides I that can be obtained from vinylstannanes with defined configurations. The vinyl stannanes will be accessed by using known chemistry reported by Lipshutz et al., Preparation of Z-Vinylstannanes via Hydrozirconation of Stannylacetylenes, Tetrahedron Lett., 33:5861-5864 (1992); Lipshutz, et al., Hydrozirconation/Transmetalation of Acetylenic Stannanes. New 1,1-Dimetallo Reagents, Inorganica Chimica Acta, 220:41-44 (1994), which utilizes a 1,1-dimetallo species as a stereodefined 1,1-vinyl dianion synthon. An exemplary synthesis is given in Scheme 9, for the precursor to a naturally occurring epothilone, and starts with a Corey-Fuchs reaction (PPh3, CBr4) of the known aldehyde 22, followed by base-induced elimination and quenching of the lithium acetylide with tributyltin chloride (Bu3SnCl) to yield alkynylstannane 23. The 1,1-dimetallo species 24 is generated by hydrozirconation of the alkynyl stannane 23 using chlorohydridozirconocene (Schwartz reagent). An aqueous quench would provide Z-vinylstannane 25a or alternatively, selective transmetalation with a higher order cuprate, followed by addition of an electrophile (MeOTf in case of epothilone B) to the resultant species provides the a-substituted vinylstannane 25b with high stereoselectivity. The Z-vinylstannanes 25a and 25b can then be transformed to the corresponding vinyl iodides I utilizing iodine with retention of configuration. 
An alternative route to the synthesis of alkynylstannane 23 (Scheme 9a) which allows for incorporation of different substituents at the C16 carbon involves the asymmetric epoxidation of secondary alcohol 18a under the Sharpless conditions using (xe2x88x92)-diisopropyl tartrate, tert-butyl hydroperoxide and titanium isopropoxide to give epoxide 19a. The alcohol function on the epoxide can be oxidized with TPAP, NMO to give ketone 20a which can be reacted with Wittig reagents containing thiazole or other aromatic/heteroaromatic rings to give the corresponding trans-olefins. The terminal epoxide in this olefin can then be opened with trimethylsilyl acetylide to give secondary alcohol 22a. The trimethylsilyl group can then be substituted for a trialkyl stannyl group on treatment of 22a with TBAF and bis-tributyltin oxide and the obtained product treated with TBSCl to give compound 23. 
The foregoing chemistries can be used for the synthesis of analog precursors as well. Such analogs are best illustrated by Formula X, wherein n3, R, R1 and R2 are as defined previously. Again all of these modified segments can then be utilized in the total synthesis of various analogs of epothilones.
In summary, although some of the vinyl iodides of the Formula X are previously reported (20,21) compounds, the method to synthesize it from the known aldehyde 22 is different from conditions reported in other total syntheses of epothilones. In addition, the above mentioned hydrozirconation reactions provide precise control over the geometry of the C12-C13 olefin bond. Also the use of other electrophiles in the transmetalation reaction with the intermediate species 24 allows for the synthesis of various analogs. 
The invention also provides new synthetic routes to specific preferred embodiments of the above Formula X defined previously, in particular C12-C20 vinyl halide epothilone precursors of the formula. 
A preferred reaction Scheme 9B set forth below illustrates this aspect of the invention. Thus, the known aldehyde 1 was treated with the phosphonate 2 in presence of barium hydroxide and wet tetrahydrofuran as solvent to give the enone 3 in 75% yield. The asymmetric reduction of this enone 3 using 50 mol % of commercially available chiral catalyst (R)-2-Me-CBS-oxazaborolidine and 1.5 equivalents of borane dimethylsulfide complex in dichloromethane gave the desired alcohol 4 in 79% yield and in 95% enantiomeric excess. The completion of the segment synthesis involved the protection of the C15 alcohol as the TBS ether to provide the known compound 5 using TBSOTf (tert-butyldimethylsilyl trifluoromethanesulfonate) and 2,6-lutidine as base. The remaining steps (i.e.conversion of 5 to I) to the known vinyl iodide I have been previously reported in literature. 
An important feature of this synthesis is the ability to produce in high enantomeric excess the alcohol 4 from the enone 3, using a chiral catalyst. This largely eliminates racemates in the alcohol, thus giving significantly higher yields.
A second method to the same C12-C20 precursor relies on the use of a Noyori asymmetric hydrogenation of xcex2-ketoesters to set the required stereochemistry for the C15 stereocenter of the epothilones. This chemistry is illustrated in Scheme 9C below, and begins with the known aldehyde 6. An aldol condensation reaction of this aldehyde 6 with the enolate anion generated from ethyl acetate using lithium diisopropyl amide as base provided the beta-ketoalcohol 7 in 76% yield. This alcohol 7 was oxidized to the beta-ketoester 8 using MnO2 (manganese dioxide) in 90% yield. The Noyori asymmetric reduction of the beta-ketoester 8 provides the chiral alcohol 9 with desired S-stereochemistry at position 15 in 50% yield and 83% enantiomeric excess. This alcohol 9 is protected as its TBS (tert-butyldimethylsilyl) ether 10 using TBSOTf (tert-butyldimethylsilyl trifluoromethanesulfonate) and 2,6-lutidine in 72% yield. The ester functionality in 10 was then reduced to the known primary alcohol 11 using DIBAL-H. The final stages in the synthesis are similar to those reported in Scheme 1 and the same conversions have been reported previously in the literature. 
Two other epothilone derivatives of special interest maybe synthesized in accordance with the invention. In one such derivative the lactone functional group is replaced with an ether functionality and in the other a lactam functionality is used in lieu of the lactone functional group. Thus in the first derivative, and referring to Formula A, X is O, Y is H2, n1, n2, and n3 are 1, R is 2-methyl-thiazol-4-yl, R1 is methyl, R2 is H or methyl, and R3, R4, R5 and R6 are methyl. In the second derivative, the only change is that X is NH and Y is O. These could be synthesized by the reaction sequences shown in Schemes 10 and 11. Thus selective deprotection at C1 by camphorsulfonic acid (CSA) (Scheme 10), formation of the mesylate derivative of the corresponding primary alcohol, selective deprotection of the C15 TBS ether and base-induced cyclic ether formation should provide compounds 26xe2x80x2. Again, the final stages in the synthesis would involve the deprotection of both the TBS groups from the macrolides (TFA, CH2Cl2) and the diastereoselective epoxidation of the C12-C13 double bond with epoxidizing agents such as dimethyldioxirane to give the ether derivatives 29 and 30.
For the lactam formation (Scheme 11) again compound 26 could be selectively deprotected at C-1 followed by sequential oxidation of the primary alcohol first under Swem conditions followed by NaClO2xe2x80x94NaH2PO4 would furnish the known acids. These known acids can be converted to their allyl esters and then the TBS ether at C15 can be deprotected selectively. Mitsunobu inversion of these alcohols and azide formation via the corresponding mesylates will provide the azides with the correct stereochemistry at C15. Reduction of the azides (PPh3, H2O) followed by salt formation of the amine will provide 32. Deprotection of the allyl esters (Pd(PPh3)4, base) followed by macrolactamization (HBTU) will provide the lactams 33. Again, deprotection of both the TBS groups from the macrolides (TFA, CH2Cl2) and the diastereoselective epoxidation of the C12-C13 double bond with epoxidizing agents such as dimethyldioxirane would give the lactam derivatives 34 and 35. 
Representative C4-C8 cycloalkyl, substituted and unsubstituted aromatic and heteroaromatic groups, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, C1-C10 alkoxy groups, and heterocyclic groups useful in the formation of epothilone analogs are set forth below.
C4-C8 cycloalkyl groups: cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
Substituted and unsubstituted aromatic groups: phenyl, phenyl groups substituted at any position with C1-C4 straight or branched chain alkyls, C1-C4 alkoxy groups, halogens, amines, amides, azides, sulfides, carboxylic acids and their derivatives, and hydroxides.
Substituted and unsubstituted heteroaromatic groups: thiazoles, pyrroles, furans, thiophenes, oxazoles and pyridines, and imidazoles.
C1-C10 straight and branched chain alkyl groups: methyl, ethyl, propyl, butyl, isopropyl, isobutyl, isopentyl, octyl, nonyl, and t-butyl.
Substituted and unsubstituted benzyl groups: benzyl, benzyl groups substituted at any position with C1-C4 straight or branched chain alkyls, C1-C4 alkoxy groups, halogens, amines, amides, azides, sulfides, carboxylic acids and their derivatives, and hydroxides.
C1-C10 alkoxy groups: methoxy, ethoxy, propoxy, butoxy, isopropoxy, t-butoxy, and nonoxy.
Heterocyclic groups: piperidines, furans, pyrroles, oxazolines, and thiophenes.
The following examples set forth various syntheses of the type described previously. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.